Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. and worldwide with enormous health and economic costs. The mTORC1-S6 kinase 1 (S6K1) axis drives anabolic pathways determining obesity. We recently identified glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to mouse adiposity. Insulin-stimulated EPRS phosphorylation at Ser999 by S6K1 in adipocytes induces its binding to fatty acid transport protein 1 (FATP1) and translocation to the plasma membrane to increase long-chain fatty acid (LCFA) uptake. Recent studies reveal that phosphorylation of S6K1 by cyclin-dependent kinase 5 (Cdk5) at Ser424 and Ser429 in the S6K1 C-terminus are required for phosphorylation of EPRS, but not for canonical substrates such as RPS6. This unexpected finding indicates that embedded in S6K1 is a target-selective phospho-code in which combinatorial phospho-site phosphorylation determines kinase targets. To identify additional targets of multi-phosphorylated S6K1 (termed S6K1*) but not mTORC1-activated S6K1, we transfected HEK cells with S6K1 bearing phospho-mimetic mutations at the 3 phospho-sites, or wild-type S6K1 cDNA. Three new S6K1* targets were identified by mass spectrometry and validated in adipocytes ? coenzyme A synthase (COASY), cortactin, and lipocalin 2. Importantly, all are implicated in adipocyte lipid metabolism: P-EPRS transports FATP1 to the plasma membrane for increased LCFA uptake; COASY catalyzes the final two steps of synthesis of coenzyme A, required for LCFA activation; lipocalin 2 increases LCFA ?-oxidation and insulin resistance; and cortactin is required for insulin-stimulated transport of Glut4-containing vesicles to plasma membranes. We propose that S6K1* directs an adipocyte lipid metabolon, and is a major contributor to obesity-related phenotypes driven by the mTORC1-S6K1 axis. We will test this hypothesis by pursuit of 3 Specific Aims: In Aim 1 we determine S6K1*/target docking domains. By mass spectrometry and site-directed mutation analysis, we will determine specific S6K1*-directed phosphorylation sites in the targets. In Aim 2 we determine the function of phosphorylated S6K1* targets in adipocyte lipid metabolism. We will determine the mechanism of insulin-stimulated transport and binding of P-EPRS to the adipocyte plasma membrane; the role of phosphorylation in COASY catalytic activity and localization; whether P-cortactin transports P-EPRS/FATP1-containing vesicles to the plasma membrane; and extracellular secretion and intracellular localization of P-lipocalin 2, and its role in LCFA oxidation. In Aim 3 we elucidate In vivo role of S6K1* in lipid metabolism and obesity. We will determine the effect of diet-induced obesity on the S6K1* activation pathway and on target phosphorylation in mice. Taking advantage of our new mouse model (generated by Crispr-Cas9 technology) bearing a Ser429-to-Ala mutation in Rps6kb1 (mouse gene encoding S6K1) that lack S6K1* activity, while retaining canonical S6K1 activity, we will test the role of S6K1* in target phosphorylation in vivo, in lipid metabolism, and in diet-induced obesity.